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Assembly Related Protocols
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Revision as of 03:17, 2 August 2008
Newest EcoRI/BamHI Transfer method (7/31/08)
Digestion:
- 0.5ul plasmid in entry vector
- 0.5ul EcoRI
- 0.5ul BamHI
- 1ul NEB2 + ATP buffer
- 7.5ul of water
- incubate the digestion for 1hr. in the thermocycler, and 20 min for heat kill.
No purification step.
Ligation:
- 1ul of dilute assembly vector (or 0.5ul if undiluted)
- 0.5ul ligase
Transform. (same as usual)
Transfers from Miniprepped DNA
(already in correct vectors - transfers into L/R cells)
In 0.5 ml tube, combine:
- 1ul plasmid dna
- 30ul of cells (L/R) + 30 KCM (no water)
Take the reaction, and:
- sit in ice for 10 min
- heat shock for 90 sec
- sit another 2 min ice
Then, incubate your tubes for 5 minutes (No rescuing is necessary)
Next, prepare plates (double antibiotics) and tubes with media (LB + antibiotics)
- Take 15ul of rxn. Spot it onto a plate (no spreading required)
- Take the remaining 15ul into a pipette tip. Drop the entire tip into your tube of media.
- Let plates and tubes sit overnight.
- Miniprep the next day.
1-2-3 Assembly
Set up the digestion:
- 1uL lefty plasmid(methylated)
- 1uL righty plasmid (methylated)
- 1uL NEB2+ATP
- 0.3 uL XhoI
- 0.3 uL BglII
- 0.3 uL BamHI
- 6.1uL Water
Heat kill at 65 for 20 min
Add 0.3uL Ligase
Transform, rescue, plate on dual antibiotic plates
